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IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
http://dx.doi.org/10.4110/in.2012.12.1.18
pISSN 1598-2629 eISSN 2092-6685ORIGINAL ARTICLE
18
Received on December 23, 2011. Revised on January 13, 2012. Accepted on January 30, 2012.CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial
License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribu-tion, and reproduction in any medium, provided the original work is properly cited.
*Corresponding Author. Jae Seung Kang, Tel: 82-2-740-8132; Fax: 82-2-741-8202; E-mail: genius29@snu.ac.kr, Wang Jae Lee, Tel: 82-2-740-8208; Fax: 82-2-745-9528; E-mail: kinglee@snu.ac.kr
†The authors are equally contributed to this work.
Keywords: Vitamin C insufficiency, Gulo−/− mice, In vivo kinetics
The Analysis of Vitamin C Concentration in Organs of Gulo−/− Mice Upon Vitamin C WithdrawalHyemin Kim1†, Seyeon Bae1†, Yeonsil Yu1, Yejin Kim1, Hang-Rae Kim1, Young-il Hwang1, Jae Seung Kang1,2* and Wang Jae Lee1*1Department of Anatomy, 2Institutes of Complementary and Integrative Medicine, Medical Research Center, Seoul National University, Seoul 110-799, Korea
Background: Vitamin C is an essential nutrient for maintain-ing human life. Vitamin C insufficiency in the plasma is closely related with the development of scurvy. However, in vivo kinetics of vitamin C regarding its storage and con-sumption is still largely unknown. Methods: We used Gulo−/− mice, which cannot synthesize vitamin C like human. Vitamin C level in plasma and organs from Gulo−/− mice was examined, and it compared with the level of wild-type mice during 5 weeks. Results: The significant weight loss of Gulo−/− mice was shown at 3 weeks after vi-tamin C withdrawal. However, there was no differences be-tween wild-type and vitamin C-supplemented Gulo−/− mice (3.3 g/L in drinking water). The concentration of vita-min C in plasma and organs was significantly decreased at 1 week after vitamin C withdrawal. Vitamin C is preferentially deposited in adrenal gland, lymph node, lung, and brain. There were no significant changes in the numbers and CD4/CD8 ratio of splenocytes in Gulo−/− mice with vita-min C withdrawal for 4 weeks. And the architecture of spleen in Gulo−/− mice was disrupted at 5 weeks after vita-min C withdrawal. Conclusion: The vitamin C level of Gulo−/− mice was considerably decreased from 1 week af-ter vitamin C withdrawal. Vitamin C is preferentially stored in some organs such as brain, adrenal gland and lung.[Immune Network 2012;12(1):18-26]
INTRODUCTION
Vitamin C is a co-factor of some enzymes such as dopamine-
β-hydroxylase and collagen synthase which are essential for
the life (1-3). That is to say, vitamin C insufficiency affects
severe defects on cardiac function and skeletal systems due
to the deficiency on the production of hormones and collagen
(4,5). In addition, vitamin C plays an important role on the
defense system against viral infection and the development
of cancer (6-8). Even though there are still the arguments re-
garding the anti-viral and anti-tumor activity of vitamin C in
vivo, lots of experiment in vitro showed that vitamin C is one
of the effective nutrients for the prevention of tumor develop-
ment and cancer therapy.
It is known that vitamin C (L-ascorbic acid) is synthesized
from glucose, during the glycolytic pathway and L-gulono-
lactone-γ-oxidase (gulo) is one of the essential enzymes for
the synthesis of vitamin C, especially conversion of L-gulono-
lactone to L-ascorbic acid (9,10). In the case of human being
and some primates, a mutation of the gene encoded gulo is
considered as the reason of the defect on the production of
vitamin C (11). However, most of experimental animals could
produce vitamin C by themselves, except guinea pig. Gulo−/−
mice were generated and used for the investigation of the
effect of vitamin C on the prevention of the formation of athe-
rosclerotic plaque upon vitamin C insufficiency (12). There-
fore, the limitation of in vivo experiments about the effect
In vivo Kinetics of Vitamin C in Gulo−/−MiceHyemin Kim, et al.
19IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
of vitamin C on the prevention or facilitation of development
of diseases upon vitamin C supplementation or withdrawal
has been overcome.
Regarding in vivo vitamin C pharmacokinetics, it was re-
ported that plasma vitamin C concentration reaches the peak
concentration at 2∼3 hrs after administration (13). When vi-
tamin C is administered via intravenous injection, its concen-
tration in serum is 5∼6 times higher than that of oral admin-
istration, and it is drastically decreased at 6 hrs after admini-
stration. According to the report by Harrison et al. (14), vita-
min C is preferentially deposited in brain (4∼10 mM), adre-
nal gland (2∼10 mM), liver (0.8∼1 mM) and cerebrospinal
fluids (CSF; 0.2∼0.4 mM). However, the reason why such
organs contain the high concentration of vitamin C is still
largely unknown. Moreover, in vivo kinetics of vitamin C in
organs under vitamin C insufficient condition has not been
investigated yet. Therefore, we examined the changes of vita-
min C concentration in organs of Gulo−/− mice upon vitamin
C withdrawal.
MATERIALS AND METHODS
MiceGulo−/−
mice were obtained from the Mutant Mouse
Regional Resource Center (University of California, Davis,
USA). C57BL/6 wild-type and the Gulo−/− mice were main-
tained in a specific pathogen-free condition at an animal fa-
cility in the Seoul National University College of Medicine.
Male Gulo−/− mice (4∼5 weeks old) were maintained for
5 weeks with or without vitamin C (3.3 g/L or 0.33 g/L,
Sodium L-ascorbate, Sigma, St. Louis, MO, USA) supple-
mentation in drinking water. The animal use protocol for the
experiment (approved No. SNU-080624-3 and SNU-100428-3)
was reviewed and approved by the Ethics Committee of the
Seoul National University.
Sample preparationDuring the experimental periods for 5 weeks, plasma and or-
gans were collected from wild-type and Gulo−/− mice with
or without vitamin C supplementation at every week. Tissues
were quickly frozen in liquid nitrogen, and then stored at
−70oC until use. After weighting, tissues were homogenized
with TissueLyser II (Qiagen, Germany) in phosphate buffered
saline (PBS). The homogenates were centrifuged with 14,000
rpm at 4oC for 30 min, and the supernatants were used for
vitamin C measurement.
Measurement of vitamin C concentrationPlasma and tissue homogenates were diluted in PBS. Vitamin
C (ascorbic acid, AA) converted into dehydroascorbic acid
(DHA), an oxidized form of ascorbic acid, and then the con-
centration of total DHA was measured by using a colorimetric
microtiter plate assay kit (Immundiagnostik AG, Germany) ac-
cording to the manufacturer’s instructions. The final concen-
tration of vitamin C in each tissue was normalized to tissue
weight.
Isolation of splenocytes and flow cytometrySpleen was removed and placed into cold washing media,
which is RPMI media containing 10% heat-inactivated fetal
bovine serum, 100 U/ml penicillin, and 100μg/ml streptomy-
cin (GIBCO, Carlsbad, CA, USA). Spleen was homogenized
by passing through 70μm nylon mesh (BD Bioscience, San
Jose, CA, USA) and centrifuged at 600 g for 10 min. The pel-
let was re-suspended in red blood cell lysis buffer (Sigma,
St. Louis, MO, USA), and washed with washing media. The
isolated splenocytes were stained with trypan blue (GIBCO,
Carlsbad, CA, USA), and countered. Freshly isolated spleno-
cytes were resuspended in FACS buffer containing 0.5% BSA
and blocked at 4oC for 10 min with Fc blocking reagent
(Miltenyi Biotec GmbH, Germany). Then, cells were stained
with anti-CD4 and anti-CD8 antibodies (BD Bioscience, San
Jose, CA, USA) on ice for 30 min and washed twice with
FACS buffer. Cells were analyzed by FACS Calibur (BD
Bioscience, San Jose, CA, USA). FlowJo software (Tree Star,
Ashland, OR, USA) was used for the data analysis.
Histological examinationSpleens were freshly excised, and fixed in 4% paraformal-
dehyde. The paraffin-embedded sections (5μm thickness)
were deparaffinized with xylene and hydrated by alcohol
series. Then, sections sere stained with hematoxylin and eo-
sin (H&E, Sigma, St. Louis, MO, USA) according to the manu-
facturer’s instructions. After mounting, stained sections were
viewed with inverted light microscopy (Olympus, Center
Valley, PA, USA).
Statistical analysisData were expressed as mean±S.D. of each group in in-
dependent experiments. For comparison of three or more
groups, data were analyzed by one-way analysis of variance
(ANOVA), followed by Newman-Keuls multiple comparison
test. A value of p<0.05 was considered to be statistically
In vivo Kinetics of Vitamin C in Gulo−/− MiceHyemin Kim, et al.
20 IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
Figure 1. Loss of weight and de-crease of plasma vitamin C con-centration in Gulo−/− mice upon vitamin C withdrawal. (A) Pheno-type of wild-type (WT) and Gulo−/−mice (KO) with vitamin C with-drawal for 5 weeks. (B) Changes of body weight upon vitamin C with-drawal were followed for 5 weeks (n=10). (C) The concentration of vitamin C of WT and Gulo−/− mice in plasma (n=4∼10). ***p<0.001 vs. vitamin C- supplemented Gulo−/−mice (KO+VC, 3.3 g/L).
significant. Statistical tests were carried out using GraphPad
InStat (GraphPad Software, San Diego, CA, USA).
RESULTS
Loss of weight and decrease of plasma vitamin C con-centration in Gulo−/− mice upon vitamin C withdrawalWe first investigated the phenotypic changes of Gulo−/−
mice
upon vitamin C withdrawal for 5 weeks. A skeletal change
was observed from Gulo−/− mice without supplementation
of vitamin C for 5 weeks (Fig. 1A). We found the decreased
movement of Gulo−/− mice upon vitamin C withdrawal (data
not shown). As shown in Fig. 1B, weight loss began at 3
weeks after vitamin C withdrawal and aggravated with
weeks. We next compared the plasma concentration of vita-
min C in four experimental groups; wild-type, Gulo−/− mice
with vitamin C supplementation (3.3 g/L or 0.33 g/L), and
Gulo−/− mice without vitamin C supplementation. There was
no difference between wild-type and Gulo−/− mice supple-
mented with 3.3 g/L concentration of vitamin C. However,
remarkable decrease of vitamin C concentration to sub-scurvy
levels (less that 30μM) was observed at 1 week after vitamin
C withdrawal and it lasted for 5 weeks (Fig. 1C). In addition,
plasma levels of vitamin C in Gulo−/− mice supplemented
with 0.33 g/L of vitamin C was compared with that of
wild-type and Gulo−/− mice supplemented with 3.3 g/L of
vitamin C. As a result, it levels in Gulo−/− mice supple-
mented with 0.33 g/L could not reach at the concentration
in wild-type and Gulo−/− mice supplemented with 3.3 g/L
of vitamin C. It suggests that the optimal concentration of vi-
tamin C is 3.3 g/L for the examination of physiological effects
of vitamin C in vivo.
The changes of vitamin C concentration in gastro-intestinal organsIn general, vitamin C uptake mainly occurred in gastro-
intestinal tracts via the vitamin C-specific transporters, sodium
dependent vitamin C transporter (SVCT)-1 and -2 (15).
Therefore, the concentration of vitamin C in stomach, large
intestine and small intestine upon vitamin C withdrawal were
examined. The concentration of vitamin C in large and small
intestine was higher than stomach (Fig. 2A-C). In addition,
it decreased at 1 week after vitamin C withdrawal and it last-
ed for 5 weeks. As we described, vitamin C is a derivative
of glucose and liver is the site for glycolysis and gluconeo-
genesis (16). Therefore we also examined the concentration
In vivo Kinetics of Vitamin C in Gulo−/−MiceHyemin Kim, et al.
21IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
Figure 2. The changes of vitamin C concentration in gastrointestinal or-gans. The concentration of vitamin C of WT and Gulo−/− mice in (A) large intestine, (B) small intestine, (C) stomach and (D) liver (n=4∼10). *p<0.05, **p<0.01, ***p<0.001 vs. vitamin C-supplementedGulo−/− mice (KO+VC, 3.3 g/L).
of vitamin C in liver. Relatively low concentration of vitamin
C was deposited in liver (Fig. 2D). Like a plasma levels of
vitamin C, the concentration of vitamin C in intestines, stom-
ach and liver of Gulo−/− mice supplemented with 0.33 g/L
concentration of vitamin C during the experimental periods
was examined. It was similar with the concentration in those
organs of Gulo−/− mice that is maintained without vitamin
C for 1 week.
The changes of vitamin C concentration in brain, heart and lungIt is already known that vitamin C plays an important role
in the collagen synthesis as a co-factor of collagen synthase
(17). Therefore, it is considered that vitamin C has protective
effect in brain and heart from the infarction through extensive
vascular changing. For this reason, we examined the levels
of vitamin C in brain and heart. As shown in Fig. 3A and
B, high concentration of vitamin C was concentrated in both
brain and heart. Interestingly, we found that the high concen-
tration of vitamin C was maintained in brain until the end
of experiments, approximately 1 mM at 5 weeks after vitamin
C withdrawal. It seems that huge amounts of vitamin C are
needed to protect brain from the damage by reactive oxygen
species (ROS), since vitamin C is one of the well-known an-
ti-oxidants. Based on the role of vitamin C as anti-oxidants,
the concentration in lung was subjected to be analyzed. As
we expected, high concentration of vitamin C is deposited
as much as brain, but its concentration was more rapidly de-
creased (Fig. 3C). The concentration of vitamin C in brain,
heart and lung of Gulo−/− mice supplemented with 0.33 g/L
concentration of vitamin C was also relatively lower than in
wild-type or Gulo−/− mice supplemented with 3.3 g/L of vi-
tamin C.
The changes of vitamin C concentration in adrenal gland, pancreas, testis and kidneyVitamin C is an essential factor for the production of hor-
mones (17). So, we investigated the concentration of vitamin
C in some organs related with the generation or action of
hormones. Adrenal glands, which mainly produce cortico-
steroid hormones, contain vitamin C up to 5 mM (Fig. 4A).
Even though it is also decreased at 1 week after vitamin C
withdrawal, high concentration of vitamin C was maintained
till 5 weeks after vitamin C withdrawal. Moreover, relatively
In vivo Kinetics of Vitamin C in Gulo−/− MiceHyemin Kim, et al.
22 IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
Figure 3. The changes of vitamin C concentration in brain, heart and lung. The concentration of vitamin C of WT and Gulo−/− mice in (A) brain, (B) heart, and (C) lung (n=4∼10). **p<0.01, ***p<0.001 vs. vitamin C-supplemented Gulo−/− mice (KO+VC, 3.3 g/L).
Figure 4. The changes of vitamin C concentration in adrenal gland, pancreas, testis and kidney. The concentration of vitamin C of WT and Gulo−/− mice in (A) adrenal gland, (B) pancreas, (C) testis and (D) kidney (n=4∼10). **p<0.01, ***p<0.001 vs. vitamin C-supple-mented Gulo−/− mice (KO+VC, 3.3 g/L).
In vivo Kinetics of Vitamin C in Gulo−/−MiceHyemin Kim, et al.
23IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
Figure 5. The changes of vitamin C concentration in lymph node and spleen, and the structural alteration in spleen. The concentration of vitamin C of WT and Gulo−/− mice in (A) lymph node and (B) spleen (n=4∼10). ***p<0.001 vs. vitamin C-supplemented Gulo−/− mice (KO+VC, 3.3 g/L). (C) Splenic tissues from WT, Gulo−/− mice with vitamin C (3.3 g/L) supplementationand Gulo−/− mice with vitamin C withdrawal for 3∼5 weeks were stained with H&E (×100). (D) The number of splenocytes and (E) the ratio of CD4 to CD8 T cells upon vitamin C supplementation and withdrawal.
low concentration of vitamin C was detected in pancreas, tes-
tis and kidney (Fig. 4B-D). The sudden fall of vitamin C con-
centration at 1 week after vitamin C withdrawal was also ob-
served in adrenal gland, pancreas, testis and kidney like other
organs. The concentration of vitamin C in these organs of
Gulo−/− mice supplemented with 0.33 g/L of vitamin C was
similar to that of Gulo−/− mice with vitamin C withdrawal
during the experimental periods.
The changes of vitamin C concentration in lymph node and spleenSince the most well-known function of vitamin C is immune
potentiating, we finally examined the concentration of vitamin
C in lymph node and spleen. Interestingly, we found that vi-
tamin C is deposited at lymph node as much as adrenal
glands (Fig. 5A). Even though the level was relatively low
when it compared with the vitamin C concentration in lymph
node, spleen contained high concentration of vitamin C like
heart, large and small intestines (Fig. 5B). In the case of
In vivo Kinetics of Vitamin C in Gulo−/− MiceHyemin Kim, et al.
24 IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
spleen, the alteration of splenic architecture in Gulo−/− mice
by vitamin C withdrawal for 5 weeks was found (Fig. 5C).
However, there were no significant differences in the spleno-
cyte numbers and at the ratio between CD4 and CD8 T cell
(Fig. 5D and E).
DISCUSSION
Vitamin C, glutathione, and vitamin E (α-tocopherol) are im-
portant members of intracellular anti-oxidant network and
they protect organisms from the damages induced by oxygen
free radicals, such as superoxide anion, nitric oxide and hy-
drogen peroxide. Even though it is known that anti-oxidant
activity of vitamin C is less than other two molecules, it
should be needed for the conversion of oxidized glutathione
(GSSG) to reduced glutathione (GSH) and tocopheroxyl radi-
cal, an oxidized form of vitamin E, to α-tocopherol (4,5).
It means that vitamin C is an essential factor for the main-
tenance of intracellular anti-oxidant network. Based on its an-
ti-oxidant activity, we can suppose that vitamin C is preferen-
tially accumulated in metabolically active organs, such as
brain, lung and heart. In fact, it is reported that vitamin C
uptake is mainly occurred at the endothelium of small intes-
tine and preferentially deposited in brain (4∼10 mM), adre-
nal gland (2∼10 mM), liver (0.8∼1 mM), muscle (0.4 mM)
and cerebrospinal fluids (CSF; 0.2∼0.4 mM), and 40∼60μM
of vitamin C is detected in serum and red blood cell (14).
To understand physiological mechanisms related with the
management of organs, in vivo vitamin C kinetic study re-
garding accumulation and its consumption in organs is strong-
ly needed. Therefore, we examined in vivo vitamin C kinetic
study upon vitamin C withdrawal by using Gulo−/− mice.
This is the first reports regarding the storage and consumption
of vitamin C in vivo using animals which cannot synthesize
vitamin C like human.
Since Gulo−/− mice is unable to synthesize vitamin C in vivo
like human, 0.33 g/L of vitamin C is recommended for their
maintenance (www.mmrrc.org). However, we found remark-
able decreasing vitamin C in plasma to the level of scurvy (0.33
g/L vitamin C-supplemented Gulo−/− mice: 24.88μM). When
mice were maintained with 3.3 g/L, it was almost same as WT
mice (WT: 88.41μM, 3.3 g/L vitamin C-supplemented Gulo−/−
mice: 80.90μM). Most organ concentration of vitamin C in
Gulo−/− mice supplemented with 0.33 g/L vitamin C was sim-
ilar to the levels of Gulo−/− mice with vitamin C withdrawal
for 1∼2 weeks. It suggests that 0.33 g/L is minimum amounts
of vitamin C only for the maintenance of Gulo−/− mice, but
3.3 g/L is optimal amounts for the investigation of the physio-
logical functions of vitamin C in vivo. SMP30−/−
mice are
also used as an animal model for the analysis of vitamin C
functions in vivo. For the maintenance of SMP30−/−
mice,
1.5 g/L of vitamin C/day was used, because alternative path-
way for vitamin C synthesis by using of D-glucurono-1, 4-lac-
tone is intact (18). Therefore, Gulo−/− mice is maintained
with 3.3 g/L concentration of vitamin C supplementation, and
it is the most suitable experimental model for the assessment
of in vivo vitamin C functions and its related mechanisms.
Vitamin C has a crucial role on the hydroxylation of pro-
line, which is closely related to collagen synthesis, blood ves-
sel formation, the synthesis of hormone and neurotransmitter
and immune functions (19). Therefore, it seems that weight
loss of Gulo−/− mice shown in Fig. 1B is caused by the de-
fect of the hydroxylation of proline and collagen synthesis un-
der vitamin C-insufficient condition. In relation with collagen
synthesis, there are several reports regarding the important
role of insulin-like growth factor (IGF) production (20,21).
That is to say, severe defect of the synthesis of collagen was
observed when IGF production is inhibited. Since we have
previously reported that vitamin C inhibits the proliferation
of human melanoma cell lines, SK-Mel2 via the suppression
of IGF production (22), the defect of the hydroxylation of
proline and collagen synthesis in Gulo−/− mice under vitamin
C-insufficient condition should be further investigated. In ad-
dition, the decrease of food intake is regarded as the one of
the reason for the weight loss of Gulo−/− mice. According
to the report by Odumosu (23), vitamin C supplementation
reduces weight loss of guinea pigs and its effect is synergisti-
cally up-regulated appetite when administered with serotonin.
Regarding the roles and functions of vitamin C on the an-
ti-viral and anti-tumor immunity, it is believed that vitamin
C directly increased the cytotoxic activity of natural killer
(NK) cells or antigen specific cytolytic T cells (CTLs), but it
is still controversial. It is because that most of experiments
regarding the effect of anti-viral and anti-tumor immunity
were done in vitro. Even if the experiments were done in
vivo, it was impossible to distinguish whether the anti-viral
and anti-tumor effects are induced by vitamin C supplementa-
tion, since most animals used in experiments can produce the
large amounts of vitamin C by themselves. However, we
found that the size of spleen was distinctively reduced and
the structure of spleen was considerably disrupted in Gulo−/−
mice by vitamin C withdrawal for 5 weeks. Therefore, it as-
In vivo Kinetics of Vitamin C in Gulo−/−MiceHyemin Kim, et al.
25IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012
sumes that vitamin C is essential for the maintenance of the
structure and functions of spleen. We also agree that vitamin
C can directly increase of anti-viral and anti-tumor activity of
NK cell and CTLs. Since we have previously reported that
vitamin C directly induces apoptosis of tumor via the
down-regulation of transferrin receptors and mitochondrial
membrane potential bypassing the activation of NK cells and
CTLs (24). The maintenance of the highest concentration of
vitamin C via intravenous injection is useful for immunoth-
erapy of cancer patient, since we can expect that vitamin C
maintains effector functions of immune organs and immune
cells as well as directly induces apoptosis of tumors.
ACKNOWLEDGEMENTS
This work was supported by the grants from Seoul National
University Hospital (Grant #0320110290) to Jae Seung Kang.
CONFLICTS OF INTEREST
The authors have no financial conflict of interest.
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